Metabolomics-based profiling of 4 avocado varieties using HPLC–MS/MS and GC/MS and evaluation of their antidiabetic activity

Seven avocado “Persea americana” seeds belonging to 4 varieties, collected from different localities across the world, were profiled using HPLC–MS/MS and GC/MS to explore the metabolic makeup variabilities and antidiabetic potential. For the first time, 51 metabolites were tentatively-identified via HPLC–MS/MS, belonging to different classes including flavonoids, biflavonoids, naphthodianthrones, dihydrochalcones, phloroglucinols and phenolic acids while 68 un-saponified and 26 saponified compounds were identified by GC/MS analysis. The primary metabolic variabilities existing among the different varieties were revealed via GC/MS-based metabolomics assisted by unsupervised pattern recognition methods. Fatty acid accumulations were proved as competent, and varietal-discriminatory metabolites. The antidiabetic potential of the different samples was explored using in-vitro amylase and glucosidase inhibition assays, which pointed out to Gwen (KG) as the most potent antidiabetic sample. This could be attributed to its enriched content of poly-unsaturated fatty acids and polyphenolics. Molecular docking was then performed to predict the most promising phytoligands in KG variety to be posed as antidiabetic drug leads. The highest in-silico α-amylase inhibition was observed with chrysoeriol-4′-O-pentoside-7-O-rutinoside, apigenin-7-glucuronide and neoeriocitrin which might serve as potential drug leads for the discovery of new antidiabetic remedies.

www.nature.com/scientificreports/ of South Africa) and KG only. Importantly, α-santalol, the major constituent of sandalwood oil, showed efficient antioxidant and anti-hyperglycemic activity in alloxan-induced diabetic animal models 18 .
Regarding the saponified compounds, fatty acid methyl esters (FAME) were the major compounds detected among all samples especially methyl linoleate and methyl oleate. Table 3 illustrates that methyl linoleate (18:2 omega 6) was the most abundant ester among the samples. Their concentrations were in the following order: KG > RE > EH > SA > UH > MH > LH. It constituted 63.24% of the total identified compounds in KG. Therefore, it could be considered as a valuable source of healthy omega-6 fatty acid. As primary precursors of lipid mediators, both omega-6 fatty acid and omega-3 arachidonic acid methyl esters are considered important structural elements of cell membranes 19 .
Consistent with our results, a recent report by Soledad et al., 2021 demonstrated that methyl esters of linoleic, and linolenic acids were the predominant phytochemicals in Mexican avocado seeds. β-sitosterol content was significantly varied (0.61-6.89%) among the seed samples 19 . Sitosterol, stearic and linoleic acids were the most predominant compounds in the ripe fruits of Spanish avocado cultivars 20 . In contrast, Salazar-López et al., 2020 reported that stigmast-5-en-3-ol was the main phytosterol of Hass avocado 21 .
More recently, methyl lineoleate, β-sitosterol and campesterol were reported to ameliorate the glucose level in sucrose induced diabetes in rats by regulation of glucose transporter proteins and insulin receptor due to their lipophilic nature 22 . Hydrocarbons and esters were important volatile components of avocado that contributed to the quality and flavor of the fruits 21 . Monoterpene and sesquiterpene hydrocarbons were identified as minor constituents among all samples. α-elemene, was the most important sesquiterpene hydrocarbons identified in KG with the highest threshold (8.51%). To the best of our knowledge, this study represents the first comprehensive volatile profiling of the seven species of avocado seeds belonging to different geographic origins. www.nature.com/scientificreports/ Avocado varietal discrimination via PCA and HCA analysis of GC/MS data matrix. Owing to the complexity of the acquired data, multivariate data analysis, viz. HCA and PCA were performed to define both similarities and differences amongst the studied avocado seed samples. The metabolic variability of the different avocado seed varieties was characterized using untargeted GC/MS based metabolomics assisted by multivariate data analyses. Data matrices obtained from the combined un-saponified and saponified seeds metabolites were employed. PCA of the combined GC/MS datasets produced a model ( Fig. 1) prescribed by PC1/PC2 accounting for 66% of the variance in compositional makeup among the different seed varieties. Score plot (Fig. 1A) revealed the general structure of the dataset and the samples grouping where Pinkerton variety represented by "SA" sample obtained from South Africa was located distant from the other samples. The most influential metabolites responsible for this separation were revealed from the PCA loading plot (Fig. 1B) as being (Z)-7-hexadecenoic acid and heptadecanoic acid, as implied by their high factor loadings over the positive side of PC1, which were more abundant in "SA" sample. Gwen variety "KG sample obtained from Kenya", located in the upper right side of the PCA score, was shown to be the most enriched in 9,12-octadecadienoic acid (linoleic acid). However, the four Hass varieties "EH, LH, UH & MH" obtained from Egypt, Lebanon, Morocco, and USA clustered close to each other and found more abundant in hexadecanoic (palmitic acid) and 9-octadecenoic acids.  www.nature.com/scientificreports/ HCA, another unsupervised pattern recognition method with different graphical presentation revealed a comparable varietal classification to PCA. HCA-derived dendrogram (Fig. 1C) depicted three clusters (I-III), in which Pinkerton variety "SA" was clustered in a separate cluster exhibiting a long distance compared with the other samples on the cluster scale. Cluster II included 2 Hass varieties i.e., "LH and MH" avocado samples obtained from Lebanon and Morocco while cluster III included Gwen variety "KG sample from Kenya" and the other sub-tree contained Reed variety "RE sample from Egypt" and the other two Hass samples "UH and EH collected from USA and Egypt" indicating their comparable fatty acid profile.
From the PCA & HCA analyses, it can be concluded that the variabilities in fatty acid accumulation in the studied seed samples obtained from a wide regional divergence were efficient in the discrimination between the different seed varieties.

Antidiabetic activity of avocado seeds. Diabetes is a chronic metabolic syndrome and is considered
a life-threatening disease characterized by the inability of the pancreas to secrete insulin or insulin resistance. Uncontrolled hyperglycemia is directly associated with a high risk of diabetic related complications such ischemic heart disease, stroke, and diabetic retinopathy. According to a recent estimate by the World Health Organization (WHO), more than 422 million patients suffered from diabetes worldwide in 2014 and the prevalence of the disease threatening will increase dramatically by 2040 to exceed 642 million 23 .
α-glycosidase and α-amylase are the key enzymes which play an essential role in the dietary digestion of carbohydrates. Enzyme inhibitors were believed to be the most effective strategy for the management of diabetes and reducing its complications. Unfortunately, acarbose, the first approved enzyme inhibitor suffered from Table 3. Relative percentage of the saponified compounds in the studied avocado seed extracts using GC/MS. The separation was established on TG-5MS column (30 m × 0.25 mm i.d., 0.25 μm film thickness). The main compounds for each species are underlined and bolded. www.nature.com/scientificreports/ unexpected side-effects such as stomach-ache, flatulence, and diarrhea 24 . Recently, the trends toward natural phytoconstituents as enzyme inhibitors have gained a special interest in many scientific studies over the synthetic hypoglycemic agents 23,24 . As shown in Table S2, Gwen avocado (KG) showed the highest inhibitory activity against both α-glycosidase and α-amylase with IC 50 of 55.07 ± 2.48 and 95.3 ± 2.02 µg/ml, respectively, compared to acarbose. On the other hand, Egyptian reed (RE) showed the lowest glucosidase inhibitory activity, with almost no significant activity against amylase. Our results are shedding the light on understanding the vital role of polyunsaturated fatty acids such as linoleic acid, and its methyl ester as antidiabetic compounds identified in a high concentration in KG (ca. 63.24%) of the saponified compounds. In agreement with a recent study of Smorowskaet al., 2021 that highlighted the potential activity of oleic and linoleic fatty acids rich fraction of blue corn extract as α-amylase inhibitor 25 .
In the same line, β-sitosterol identified in Syzygium cumini L. leaves was responsible for the antidiabetic activity 26 . α-Humulene, a natural monocyclic sesquiterpene, and 9,12-octadecadienoic acid methyl ester as well as linoleic acid esters, identified in KG extract, were previously reported as natural antioxidants with antidiabetic and neuroprotective activities in the essential oil of Elaeagnus umbellata Thunb. fruits 27 .
In addition, epigallocatechin-3-gallate (EGCG) was identified by HPLC-MS/MS analysis as a unique component in KG extract which may contribute to its potential anti-diabetic activity. In a randomized placebocontrolled clinical study, catechins of green tea especially EGCG demonstrated a non-competitive α-glucosidase inhibitory activity higher than acarbose 28 . Recently, many polyphenolic compounds have been detected in cereals as quercetin, kaempferol, luteolin, naringenin and apigenin glycosides, which were reported as potent inhibitors of both amylase and glucosidase enzymes 29 .
Ultimately, the synergistic activity of both major and minor bioactive metabolites in KG was responsible for its potential activity as an amylase and glucosidase inhibitors.
Virtual screening of avocado phytoligands as potential α-amylase inhibitors. Sixteen compounds were docked into the active site of α-amylase, a crucial enzyme involved in diabetes ( Fig. 2 and Table 4). The docking simulations results showed that apigenin-7-O-rhamnoglucoside exhibited the highest α-amylase inhibitory activity, possessing a binding score of − 20.58 kcal/mol which was higher than that of acarbose (− 19.40 kcal/mol), followed by naringenin which exhibited a binding score of -20.06 Kcal/mol. Other compounds displayed a relatively low binding energy compared to the reference drug, acarbose. These include catechin gallate, chrysoeriol 7-rutinoside, eriodictyol-7-O-neohesperidoside, and epigallocatechin gallate. They also shared similar interactions like those shown from acarbose such as Glu 200 (Hydrogen bonding), Glu 207 www.nature.com/scientificreports/ (Hydrogen bonding), and Asn 174 (Hydrogen bonding). A moderate inhibitory activity on amylase enzyme was observed by amentoflavone, caffeic acid, and 4-caffeoylquinic acid, (Fig. 2 and Table 4). Accordingly, 15 phytoligands Fig. 2 showed strong interactions with the active site residues of the α-amylase enzyme. As a result, they might be considered as the potential drug candidates for the discovery of novel α-amylase inhibitors after they are screened for their safety profile and efficacy via in-vivo and clinical studies.  Table 4.

Conclusion
Agro-industrial by-products represent a global challenge due to their impact on economic, social, and environmental sectors. The green recycling of these by-products poses a crucial strategy to fight diseases. In our study, compositional differences existing among the investigated avocado varieties were assessed using GC/MS and HPLC-MS/MS analysis assisted by unsupervised pattern recognition tools. Gwen seeds exhibited the highest antidiabetic potential against two diabetic marker enzymes, i.e., α-amylase and α-glucosidase, compared to acarbose drug. This activity could be attributed to the presence of high levels of polyunsaturated fatty acids as well as several polyphenolic compounds as revealed by the molecular docking study. Our findings revealed that Gwen's variety of avocado represents a promising, low cost, and readily available natural source of bioactives to be used as an antidiabetic remedy. Further studies are still required for the successful commercial use of Gwen avocado by-products as a sustainable source of antidiabetic metabolites. Moreover, comprehensive studies are still required to investigate the effect of fruit ripening on the metabolic profile of the seeds, especially Gwen cultivar.

Material and methods
Plant material. Seven well characterized samples of avocado fruits were purchased in June 2016 from different geographical localities across the world. Three avocado varieties, i.e., Reed, Pinkerton and Gwen were obtained from Egypt, South Africa, and Kenya, respectively. In addition to, Hass cultivar was obtained from Egypt, Lebanon, USA and Morocco. They were selected at the full ripening stage. All fruits were collected and identified with permission from the Agriculture Research Center, Giza, Egypt at "9 Cairo University Road, Giza District, Giza Governorate". The fruit collection process has been established according to the national guidelines. The plant material was kindly validated by Dr. Essam Abel Karim (Tropical Fruit Research Institute, Giza, Egypt).
The voucher specimens were deposited at Faculty of Pharmacy Herbarium, Cairo University, under the identification numbers listed in Table S1. The seeds were separated from their fruit and weighted using a sensitive balance. The variation in weights was listed in Table S1. They were dried at 50 °C and grounded to fine powder. The powdered seeds (5 gm) were defatted using hexane (3 × 150 ml), which were then extracted with methanol

High-performance liquid chromatography-tandem mass spectrometer (HPLC-MS/MS). The
LC system was Thermo Finnigan (Thermo Electron Corporation, Waltham, MA, USA) equipped with a reversed-phase column (Zorbax Eclipse XDB-C18, 4.6 × 150 mm, 3.5 µm, Agilent, Santa Clara, CA, USA) as described before 30 . Briefly, LC instrument was connected to the mass spectrometer (LCQ-Duo ion trap) with an electrospray ionization source. Water and acetonitrile (ACN) with 0.1% formic acid in both solvents were used to create a gradient flow of the mobile phase. A gradual increment of ACN, from 5 to 30% for 60 min and increased to 90% in the last 30 min, was performed at a flow rate of 1 ml/min with a split ratio of 1:1. The full scan acquisition method was established, and the spectra scan range was acquired at m/z 50-2000 in the negative mode. The browser X-calibur software (X-calibur ™ 2.0.7, Thermo Fisher Scientific, Waltham, Ma, USA) was used to evaluate the data. Table 4. Docking simulations results of the studied phytoligands identified by GC/MS and HPLC-MS/MS analysis, respectively using acarbose as a co-ligand. *The best ligand-receptor complex binding free energy at RMSD < 2 .